Exploring the antimicrobial, antioxidant, anticancer, biocompatibility, and larvicidal activities of selenium nanoparticles fabricated by endophytic fungal strain Penicillium verhagenii

Herein, four endophytic fungal strains living in healthy roots of garlic were used to produce selenium nanoparticles (Se-NPs) via green synthesis. Penicillium verhagenii was found to be the most efficient Se-NPs producer with a ruby red color that showed maximum surface plasmon resonance at 270 nm. The as-formed Se-NPs were crystalline, spherical, and well-arranged without aggregation, and ranged from 25 to 75 nm in size with a zeta potential value of −32 mV, indicating high stability. Concentration-dependent biomedical activities of the P. verhagenii-based Se-NPs were observed, including promising antimicrobial activity against different pathogens (Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, Candida albicans, C. glabrata, C. tropicalis, and C. parapsilosis) with minimum inhibitory concentration (MIC) of 12.5–100 µg mL–1. The biosynthesized Se-NPs showed high antioxidant activity with DPPH-scavenging percentages of 86.8 ± 0.6% at a concentration of 1000 µg mL–1 and decreased to 19.3 ± 4.5% at 1.95 µg mL–1. Interestingly, the Se-NPs also showed anticancer activity against PC3 and MCF7 cell lines with IC50 of 225.7 ± 3.6 and 283.8 ± 7.5 µg mL–1, respectively while it is remaining biocompatible with normal WI38 and Vero cell lines. Additionally, the green synthesized Se-NPs were effective against instar larvae of a medical insect, Aedes albopictus with maximum mortality of 85.1 ± 3.1, 67.2 ± 1.2, 62.10 ± 1.4, and 51.0 ± 1.0% at a concentration of 50 µg mL–1 for I, II, III, and IV-instar larva, respectively. These data highlight the efficacy of endophytic fungal strains for cost-effective and eco-friendly Se-NPs synthesis with different applications.


Results and discussion
Isolation and primary identification of root endophytic fungi. Endophytic fungi are one of the significant organisms' having a wide range of biomedical and biotechnological applications including the production of active metabolites, utilization as biofertilizers due to their plant growth-promoting activity, phytopathogenic control, and green synthesis of nanomaterials of unique properties 17,18 . In the current study, the roots of garlic were used as the source for the isolation of endophytic fungal strains. Four fungal isolates designated as AR.1-AR.4 were obtained from collected healthy roots. These strains were identified using traditional methods based on cultural and microscopic characterizations as Penicillium sp. (AR.1), Aspergillus niger (AR.2), Alternaria alternata (AR. 3), and Penicillium sp. (AR.4) (Fig. 1). In a similar study, twelve endophytic fungal strains belonging to Aspergillus spp., Alternaria spp., Penicillium spp., Cladosporium sp., Chaetomium sp., and Fusarium sp. were isolated from Allium sativum 19 .
Screening for Se-NPs biosynthesis and identification of the most potent endophytic fungal strain using molecular analysis. Recently, researchers tended to produce new active compounds with an eco-friendly approach. Among these active compounds, nanomaterials possess high activity in various fields of agriculture, medicine, and industry 20 . The synthesis of these materials by green approaches is preferred to avoid the negative impacts originating from chemical and physical approaches 21 . Endophytic microbes including fungi, bacteria, and actinomycetes are considered promising sources for the green synthesis of nanomaterials due to the secretion of huge active metabolites that are used as reducing and capping agents 8 . In the current study, the efficacy of endophytic fungal strains in the synthesis of Se-NPs was investigated. Once adding metal precursor (Na 2 SO 3 ) to the fungal biomass filtrate, the color was changed from colorless to red color which increased gradually, indicating the formation of Se 0 due to the reduction of SeO 3 2-. The previous mixture remained for 24 h in dark conditions to confirm the complete reduction of metal and no further color change. Recently, the complete reduction of Na 2 SO 3 by the action of metabolites secreted by the endophytic fungal strain, www.nature.com/scientificreports/ P. crustosum, whereas the Se 0 form was completed after 24 h of incubation 14 . Also, the fabrication of Se-NPs through the reduction of Na 2 SO 3 using metabolites secreted by Trichoderma atroviride was observed after 24 h, and no further color change was noticed 22 . Herein, after 24 h, the absorbance of the formed color was measured to detect the maximum surface plasmon resonance (SPR). As shown, the incubation period showed a positive impact on the color intensity without any shifting in the range of SPR.  Interestingly, the maximum color intensity and the maximum SPR absorption peak were recorded for strain AR.1. The obtained results were compatible with those reported that the maximum SPR for Se-NPs synthesized by fungal strains lies in the range of 250-300 nm. For instance, among 75 endophytic fungal strains, only four strains identified as Aspergillus quadrilineatus, A. ochraceus, A. terreus, and Fusarium equiseti showed high activity for Se-NPs synthesis based on color change and maximum SPR which appeared at 265 nm 11 . Also, the maximum SPR of Se-NPs fabricated by Penicillium corylophilum and endophytic fungal strain P. crustosum was observed at 275 and 270 nm, respectively 14,23 .
According to the data of UV-Vis spectroscopy, the endophytic fungal strain designated as AR.1 was selected as the most potent strain for green synthesis of Se-NPs. This strain has undergone molecular identification based on amplification and sequencing of internal transcribed spacer (ITS) genes and was identified as Penicillium verhagenii (Fig. 3). The ITS sequence of the endophytic strain AR.1 was deposited in GenBank under the accession number of OP471232. Penicillium is composed of distinct species which are advantaged by their ability to produce various active metabolites that are used as reducing and stabilizing agents during green synthesis 24 .   www.nature.com/scientificreports/ Characterization of Se-NPs fabricated by Penicillium verhagenii strain AR.1. As mentioned, the color change followed by the detection of maximum SPR using UV-Vis spectroscopy was the first monitor for the successful formation of Se-NPs. The endophytic fungal isolate AR.1 showed the highest color intensity and absorption peak at 270 nm which corresponds to the SPR for Se-NPs. The functional groups exist in fungal biomass and their activity in reduction and stabilizing synthesized Se-NPs were investigated by Fourier transform infrared (FT-IR) analysis. As shown, the fungal biomass filtrate contained only four peaks at wavenumbers  3380, 2068, 1634, 535 cm -1 , whereas in the case of Se-NPs, the number of peaks increased to nine at wavenumbers 3400, 2880, 1565, 1415, 1380, 920, 780, 512, and 410 cm -1 (Fig. 4A). The strong and broadness peak at 3380 cm -1 could be attributed to the O-H and N-H groups of proteins and amino acids 25,26 , this peak was shifted to 3400 cm -1 at Se-NPs. The broadness peak at 2068 cm -1 is related to the carbohydrate moiety secreted by endophytic fungal strains. Moreover, the peak at 1634 cm -1 corresponds to the carbonyl (C=O) group that is overlapped with the stretching NH group of polysaccharides present in biomass filtrate 4 . This peak was shifted to 1565 cm -1 after the green synthesis of Se-NPs. A peak at 535 cm -1 in biomass filtrate could be attributed to stretching C-l of the halo compound. The presence of other peaks in the FT-IR chart of Se-NPs could be related to the interaction between metabolites in biomass filtrate with sodium selenite during the reduction and capping of as-formed Se-NPs. The medium peak at 2880 cm -1 signifies C-H stretching alkane, whereas the medium peaks in the range of 1380-1420 cm -1 could have corresponded to the bending O-H of carboxylic acid 27,28 . The peaks in the range of 400-800 cm -1 are corresponding to bending and stretching Se-O which resulted from the reaction of Se-NPs with carbonyl groups ultimately forming a coating layer around the Se-NPs surface that prevents the aggregation and agglomeration as reported previously 29 . Based on FT-IR analysis, the presence of different metabolites in fungal biomass filtrates such as proteins, polysaccharides, carbohydrates, and amino acids exhibited a crucial role in the reduction of sodium selenite to form Se-NPs followed by forming a coating that enhances the NPs stability and prevents the aggregation. The crystalline structure of as-formed Se-NPs was investigated by X-ray diffraction (XRD) analysis (Fig. 4B). As shown the XRD pattern displayed eight absorption peaks of (100),    11 . The morphological characteristics of fungal-mediate synthesized Se-NPs such as size, shape, and aggregation are principal factors that affect biological activities were investigated by transmission electron microscopy (TEM) analysis. Figures 5A and B displayed the spherical shape of synthesized Se-NPs that are well arranged without agglomeration and have sizes in the range of 25-75 nm with an average size of 44.1 ± 15.4 nm. The TEM image of Se-NPs formed at a concentration of 1.5 mM by harnessing metabolites of Acinetobacter sp. SW30 showed the successful fabrication of a spherical shape with a size of 78 nm 32 . The incorporation of NPs in different applications mainly depends on various factors such as capping agent, surface charge, shape, size, and agglomeration 21 . As size decreased, the activity increased. For instance, Se-NPs synthesized by biomass filtrate of Pantoea agglomerans showed higher antioxidant activity at smaller sizes 33 . Also, the activities of NPs are varied according to shape. For instance, Se-NPs showed high antioxidant activity for cubic shape and high antimicrobial activity for spheric shape 34 .
The size of fungal-based Se-NPs in colloidal solution was detected by dynamic light scattering (DLS). As shown, the average hydrodynamic size of synthesized Se-NPs was 91.2 nm (Fig. 5C). In the current study, the average size obtained by DLS is bigger than those obtained by TEM and XRD. This finding could be attributed to the DLS measuring hydrodynamic residue (hydrated state) whereas TEM calculates the diameter of the solid state 35 . Also, DLS is affected by coating agents and non-homogenous distribution which increases the sizes 36,37 . In a similar study, the average particle sizes of Se-NPs obtained by TEM were 15-40 nm whereas those obtained by DLS were 20-60 nm 4 which was explained by the hydrodynamic coating around the particles.
The stability of synthesized Se-NPs was investigated by Zeta potential which measures the electric charge on the NP's surface. In the current study, the fungal-based Se-NPs have a Zeta potential value of -32 mV which refers to the high stability (Fig. 5D). Similarly, the Zeta potential value of Se-NPs fabricated by aqueous extract of Carica papaya was −32 mV 38 . The stability of Se-NPs fabricated in the current study could be attributed to the presence of a negative charge which increases the negative electrostatic force between the particles and hence enhances the dispersion. Also, the stability of NPs can be classified according to Zeta potential values as follows: unstable, moderate, stable, and high stability for the values of ± 0-10, ± 10-20, ± 20-30, ˃ ± 30, respectively 39 . www.nature.com/scientificreports/ Moreover, the absence of double charges (positive and negative) and the presence of a single charge (negative) on the NPs surfaces could increase the stability as the dispersion between the particles bears the same charge to avoid aggregation. Dhanjal and Cameotra 40 reported that the presence of positive charges on the surface of some particles and negative charges on others in the same solution enhances their aggregation.
Antimicrobial activity. Most of the mortality or morbidity worldwide is caused due to microbial infectious diseases. Bacterial infections are increasing day-to-day due to the abuse of antibiotics leading to the emergence of antibiotic-resistant strains 6,41 . Further, Candida strains are opportunistic fungi and are considered the most common agent for infectious diseases such as oral candidiasis, candidemia, vaginitis, systematic infections, and cutaneous candidiasis in immunocompromised patients 42 . Therefore, it is important to construct new active compounds, safe and cost-effective to overcome these challenges. Selenium ions exhibited efficacy as antimicrobial agents and were previously used as additives to antidandruff shampoos due to their promising antifungal activity 43 . Unfortunately, they are used in little amounts due to toxicity to mammalian cells 38 . Therefore, the researchers focused on reducing their toxicity by converting them to nano-scale.
In the current study, the activity of green synthesized Se-NPs to inhibit the growth of pathogenic bacteria represented by Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus as well as different pathogenic Candida species designated as C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis were investigated by well diffusion method. Data analysis showed that the antimicrobial activity of Se-NPs was concentration-dependent. The obtained findings were compatible with the published literatures. For instance, the Se-NPs formed by the action of the metabolites in the aqueous extract of Ceropegia bulbosa displayed high antimicrobial activity against B. subtilis and E. coli at 100 µg mL -1 followed by concentrations of 75, 50, and 25 µg mL -116 . In the current study, the highest antibacterial and anti-Candida activity was recorded at 400 µg mL -1 with inhibition zones of 15.7 ± 0.6, 15.3 ± 0.6, 20.7 ± 0.7, 18.3 ± 0.6, 18.3 ± 0.6, 17.7 ± 0.5, 17.3 ± 0.5, and 16.7 ± 0.6 mm for B. subtilis, S. aureus, P. aeruginosa, E. coli, C. albicans, C. tropicalis, C. glabrata, and C. parapsilosism respectively ( Fig. 6A,B). The activity was decreased at low concentrations to 12.3 ± 0.6, 11.7 ± 0.5, 16.7 ± 0.7, 14.0 ± 0.0, 12.3 ± 0.6, 14.0 ± 0.0, 13.0 ± 1.0, and 12.7 ± 0.6 at 200 µg mL -1 of synthesized Se-NPs for the same sequence of above-mentioned test organisms. The as-formed Se-NPs by leave aqueous extract of Withania somnifera showed antibacterial activity against B. subtilis, S. aureus, and Klebsiella pneumoniae with the zone of inhibitions of 14.0 ± 0.0, 19.7 ± 0.6, and 12.0 ± 0.0 mm, respectively with no activity against E. coli 6 . Also, the Se-NPs fabricated by biomass filtrate of Penicillium corylophilum showed broad antibacterial activity against Gram-positive bacteria (B. subtilis and S. aureus) and Gram-negative bacteria (P. aeruginosa and E. coli) 23 .
The minimum inhibitory concentration (MIC) is the lowest concentration that has the efficacy to inhibit microbial growth. The choice of bioactive compounds to be incorporated into the biomedical sector is dependent on the evaluation of MIC value 44 . Herein, the synthesized Se-NPs showed varied MIC values based on the organism. For instance, the MIC value for Gram-positive bacteria was 50 µg mL -1 , whereas it was 12.5 and 25 µg mL -1 for Gram-negative bacteria, P. aeruginosa and E. coli, respectively (Fig. 6A). On the other hand, the MIC value for tested unicellular fungi were in the ranges of 50-100 µg mL -1 (Fig. 6B). Recently, the MIC value of the green synthesized Se-NPs against Candida species was varied to be in the range of 25-200 µg mL -1 according to the type of specie 14 . Also, the antibacterial activity and MIC values were varied according to the biosynthetic approach. For instance, the MIC value of Se-NPs synthesized by Aspergillus quadrilineatus and A. ochraceus against E. coli was 62.5 µg mL -1 , whereas it was 250 µg mL -1 for those fabricated by A. terreus and Fusarium equiseti 11 . This finding can be attributed to the capping agent secreted by microorganisms or plants that have a crucial role in stabilizing the nanomaterials 8,45 . Based on the obtained data, the as-formed Se-NPs by endophytic fungal strain P. verhagenii displayed high antimicrobial activity toward pathogenic bacteria and Candida spp.
The antibacterial activity of Se-NPs could be related to the cell wall structure, which in Gram-positive strains is composed of thick layers of peptidoglycan compared to thin layers in Gram-negative strains. This difference can affect the diffusion of NPs inside the cells. The thick peptidoglycan layer can prevent or delay the diffusion of Se-NPs leading to less antimicrobial activity toward Gram-positive bacteria than Gram-negative bacteria 46 . The activity toward Gram-positive bacteria can be attributed to the high electrostatic repulsion of Se-NPs toward the negative charge of lipopolysaccharide that exists in high amounts in Gram-negative bacteria than Grampositive. This lead to high deposition of Se-NPs on the surface of Gram-positive strains that ultimate in cell death 47 . Another antimicrobial mechanism of Se-NPs could be the production of reactive oxygen species (ROS) upon entry into microbial cells. These ROS such as H 2 O 2 , O 2 •-, and • OH can destroy the selective permeability function of the cytoplasmic membrane, increase the stress inside the cell, lead to inhibit DNA replication, destroy protein synthesis, and inhibit normal cell metabolism. All these dysfunctions lead to cell death 48,49 .
Interestingly, the fungal-mediated green synthesis of Se-NPs exhibited high activity against different strains of Candida. This finding could be related to its activity to destroy the sterol profile in the Candida cell wall by inhibiting the ergosterols biosynthesis pathway 50 . Also, a high accumulation of Se-NPs onto the Candida cell wall leads to the reaction of Se with sulfur-containing amino acids such as methionine and cysteine 51 . As a consequence of this interaction, the new structure which was S-Se-S was formed and modified the structure of proteins leading to the blocking of its catalytic functions 52 .
Antioxidant activity. The scavenging activity of free radicals by green synthesized Se-NPs was investigated by the DPPH method compared to the control (ascorbic acid) (Fig. 7). Data analysis displayed that the free radicals scavenging activity is a concentration-dependent nature. The activity is directly proportioned with green synthesized Se-NPs concentration. The obtained finding was compatible with published literatures that reported the scavenging activity of Se-NPs fabricated by plants, fungi, and actinomycetes was dependent on their concentrations 4,11,53 . The maximum DPPH-scavenging activity was recorded at 1000 µg mL -1 with percentages www.nature.com/scientificreports/ of 86.8 ± 0.6% compared to ascorbic acid at the same concentration which recorded a percentage of 97.3 ± 0.2% (Fig. 7).  11 . In the current study, the values of EC 50 (effective concentration for scavenging 50% of free radicals) were 28.7 ± 1.6 µg mL -1 and 5.4 ± 0.8 µg mL -1 for Se-NPs and ascorbic acid, respectively. Our data was incompatible with those recorded that the EC 50 of Se-NPs synthesized by biomass filtrate of fungal strain Monascus purpureus was 85.9 µg mL -154 , indicating the promising antioxidant activity of Se-NPs synthesized herein. Antioxidant substances or free radical scavengers are those that prohibit cell damage caused by unstable molecules or free radicals synthesized under stresses such as pathogens, contaminants, radioactive substances, toxins, etc. 55 . The main symptoms of these free radicals are rheumatism, immune dysfunction, Parkinson's, leukemia, heart attack, metabolic disorders, and respiratory failure 56 . The origin of these free radical scavengers is either natural sources such as phenolics, flavonoids, phytoestrogens, and tannins, or synthetic sources such as nanomaterials 38 . Metal and metal-oxide nanoparticles are characterized by their ability as free radical scavengers 33,56 . The activity of Se-NPs as antioxidants could be attributed to their efficacy in the upturn of selenoenzymes such as glutathione peroxidase which protect the cells from the deleterious effect of free radicals under in-vivo conditions 4,45 . Also, the antioxidants of NPs could be due to the inhibition and neutralization of  Anticancer and biocompatibility. The anticancer activity of green synthesized Se-NPs was investigated against two cancer cells designated as MCF7 and PC3, whereas the biocompatibility was assessed toward two normal cells represented as Vero and WI38. The cell viability and cellular proliferation due to Se-NPs treatment were assessed by the MTT assay method. Data analysis showed that the endophytic fungal strain-mediated green synthesis of Se-NPs has a promising anticancer activity against tested cancer cell lines in a concentration-based manner. At low concentrations (≤ 62.5 µg mL -1 ), the Se-NPs have no significant effects on the viability of cancer and normal cell lines (the cell viability in the range of 89-99% for all cells), whereas, at a concentration of 125 µg mL -1 , the viability of PC3 was 89.7 ± 0.9% compared to cancer cell lines of MCF7 (99.6 ± 3.2%) and two normal cell lines (98.4 ± 3.1% and 99.9 ± 1.2% for Vero and WI38, respectively) (Fig. 8). By increasing the Se-NPs concentration to 500 µg mL -1 , the viability was highly decreased for cancerous cell lines that reached 26.3 ± 1.8% and 8.3 ± 0.9% for MCF7 and PC3, respectively compared to the viability of normal cell lines (44.4 ± 0.7% and 43.1 ± 0.9% for Vero and WI38, respectively). Compatible with the obtained results, the activity of Phyto-synthesized Se-NPs was dependent on the concentrations against cancer cell lines of MCF-7, Caco-2, IMR-32, and normal cell line Vero 38 . Also, the antiproliferative activity of Se-NPs synthesized by harnessing metabolites of Acinetobacter sp. SW30 against 4T1, MCF7, NIH/3T3, and HEK293 were concentration-dependent manner 32 . www.nature.com/scientificreports/ Data analysis showed that the toxicity of fungal-mediated green synthesis of Se-NPs was the highest against PC3 compared to MCF7 (Fig. 8). On the other hand, the sensitivity of two normal cells toward various concentrations of Se-NPs was similar, except at 250 µg mL -1 , the viability of WI38 was decreased compared to Vero cells. The IC 50 (concentration for inhibiting 50% of cellular viability) of Se-NPs was evaluated to be 225.7 ± 3.6, 283.8 ± 7.5, 454.8 ± 29.9, and 472.8 ± 5.8 µg mL -1 for PC3, MCF7, WI38, and Vero cell lines, respectively. The obtained data reveal the target orientation of Se-NPs toward cancerous cell lines at low concentrations compared to normal cell lines. This finding confirmed the ability to integrate Se-NPs in biomedical sectors at a concentration below 300 µg mL -1 to be more active against cancer cells with negligible effects on normal cells. The minimum cytotoxic effects of synthesized Se-NPs toward normal cells could be related to the normal redox balance as reported previously 59 .
The microscopic examination of cells treated with Se-NPs showed complete or partial destruction of a monolayer of epithelial cells under high concentrations (See supplementary data, Fig. S1, S2, S3, S4). Moreover, at these concentrations, the cells tend to be rounding or grainy, shrinking, buoyancy, and decrease in the total number. These negative impacts were reduced at low concentrations, especially for normal cells. The small size is the reason for cytotoxic activity due to their efficacy to penetrate the mammalian cell membranes and interact with cell components such as proteins, nucleic acids, and amino acids leading to cellular dysfunction 60 . Moreover, the presence of Se-NPs inside the cells can increase the generation of ROS which have harmful effects on mitochondria, and ultimately to apoptotic death 14 . Shiny et al., reported that the exposure of lung carcinoma cells (A549) to silver and platinum nanoparticles leads to destroying of the cellular cytoskeleton and hence the release of specific enzymes called cytosolic LDH enzymes which are responsible for the lysis of the cells 60 .
Larvicidal activity. The use of chemical substances to control mosquito vectors had negative impacts not only on human health but also on the environment and the emergence of new resistant vectors 61 . Therefore, it is urgent to construct safe, eco-friendly, cost-effective, and rapid effect active compounds. Herein, fungal-based Se-NPs showed high mortality against the different instar larvae (I, II, III, and IV) of Aedes albopictus at various concentrations (10,20,30,40, and 50 µg mL -1 ) ( Table 1). As shown, the activity of Se-NPs against instar larvae was in a concentration-dependent manner. The obtained finding was compatible with literatures about the effect of green synthesized nanomaterials against mosquito vectors 62,63 . Data analysis showed that the highest mortality (85.1 ± 3.1%) for the I instar larva was recorded at a concentration of 50 µg mL -1 , whereas this percentage was decreased by decreasing the concentration to reach 59.0 ± 2.0% at 10 µg mL -1 . On the other hand, the as-formed Se-NPs showed high activity against IV instar larva with mortality percentages of 31.3 ± 1.5, 34.1 ± 0.0, 42.2 ± 1.0, 46.2 ± 1.6, and 51.0 ± 1.0% for concentrations of 10-50 µg mL -1 , respectively.
Analysis of variance revealed that the LC 50 and LC 90 for I, II, III, and IV instar larva of A. albopictus were (15.2, 33.6, 45.5, and 54.3 µg mL -1 ) and (132.8, 138.9, 142.4, and 180.3 µg mL -1 ), respectively (Table 1). In a similar study, the LC 50 and LC 90 of plant-based Se-NPs against instar larva of A. albopictus were in the ranges of (15.2-52.3 mg L -1 ) and (132.7-178.3 mg L -1 ), respectively 16 . Se-NPs fabricated by leaf aqueous extract of Clausena Table 1. Larvicidal activity of Se-NPs at different concentrations against instar larvae of Aedes albopictus. LC 50 (concentration for 50% mortality); LC 90 (concentration for 90% mortality); LCL (lower confidence limit); UCL (upper confidence limit); x 2 (chi-square value). Data are represented by the means of five replicates ± SE. www.nature.com/scientificreports/ dentata exhibited larvicidal activity toward Culex quinquefasciatus, Aedes Aegypti, and Anopheles stephensi with LC 50 values of 99.6, 104.1, and 240.7 mg L -1 , respectively 64 . The activity of Se-NPs toward A. albopictus could be attributed to their efficacy to penetrate the cell membrane and interact with different cellular components leading to dysfunction 16 . Also, the presence of Se-NPs inside the cell can increase the oxidative stress that is ultimate in cell death by generating toxic ROS. Moreover, Se-NPs can destroy the cells by reacting with the -SH group of amino acids or phosphorus-containing nucleic acids 65 .

Material and method
Plant sample. The roots of garlic (Allium sativum L.) from healthy plants were collected and used as a source for the isolation of fungal endophytes. Our work complies with institutional, national, and international guidelines and legislation. Garlic is common worldwide and does not need permission or licenses as the species we are working with is a cosmopolitan crop that is not at risk or endemic according to IUCN. The root samples were collected from agricultural land in El-Menofia governorate, Egypt (30° 38′ 40.9″ N 30° 56′ 49.9″ E) under permission (number: EM2/2022) from the local agricultural office in the governorate. Roots of healthy garlic plants were collected and kept in sterilized polyethylene bags before being transferred to the Laboratory using an icebox. During sample collection, five individual plants were obtained, and from each plant, three individual roots were collected.
Isolation of fungal endophytes. The collected roots are rinsed thrice with tap water to remove any adhering particles followed by rinsing with sterilized distilled H 2 O. After that, the roots were subjected to surface sterilization by immersing them into the following solutions in order: sterilized distilled H 2 O for 60 s, ethanol (70%) for 30 s, sodium hypochlorite (2.5%) for four minutes, ethanol (70%) for 30 s, and finally washing the roots with sterilized dis. H 2 O. To confirm the surface sterilization process, the sterilized dis. H 2 O from the last washing was inoculated into appropriate agar media for the growth of different microorganisms (nutrient agar for bacteria, Czapek Dox for fungi, and starch nitrate for actinomycetes). The inoculated plates were incubated at appropriate conditions and observed daily to check the growth of microbes. The absence of microbial growth in inoculated agar media indicates the success of the surface sterilization process.
The sterilized roots were cut into small segments (4 mm/segment) and ten parts per individual plant were put on the surface of the Czapek Dox agar plate (5 segments/plate) supplemented with chloramphenicol to suppress the bacterial growth. The plates were incubated at 25 ± 2 °C for 15 days and observed daily to check the appearance of fungal growth from internal plant tissues, hence picked up and re-inoculated into a new plate. The purified fungal isolates were identified based on morphological and cultural characteristics according to standard keys for Penicillium spp. 66 , Aspergillus spp. 67 , and Alternaria spp. 68 .
Checking for the activity of isolated endophytic fungi to the biosynthesis of Se-NPs. The efficacy of isolated endophytic fungal strains to act as a biocatalyst to fabricate Se-NPs was investigated. One disk (10 mm) from each fungal strain was inoculated separately in 100 mL of Czapek Dox broth media and incubated at 25 ± 2 °C for 7 days under shaking conditions (150 rpm). At the end of the incubation period, the fungal biomass was collected by filtration of inoculated broth media using Whatman No.1 and washed thrice with sterilized dis. H 2 O to remove any adhering media components. Approximately 7 g of biomass collected for each fungal strain was suspended into 100 mL dis. H 2 O and incubated at 25 ± 2 °C for 24 h followed by centrifugation to collect the biomass filtrate which was used to fabricate Se-NPs as follows: 85.5 mg of metal precursor (Na 2 SO 3 ) was dissolved in 10 mL dis. H 2 O and added to 90 mL fungal biomass to get a final concentration of 5 mM. The mixture was subjected to stirring at 40 °C for one hour and adjusted the pH at 8 using 1N NaOH, before being left at room temperature overnight under dark conditions. The conversion of color from colorless to ruby red color indicates the formation of Se-NPs followed by measuring their absorbance at a wavelength in the range of 200-700 nm to detect the maximum surface plasmon resonance (SPR) 31 . The fungal biomass filtrate without metal precursor was used as a control. The most potent fungal strain was selected due to its efficacy to form the highest ruby red color and maximum SPR value. The resultant was collected and rinsed thrice with dis. H 2 O before being undergone oven-dry at 200 °C for 4 h.

Molecular identification of the most potent fungal isolate.
The selected most potent endophytic fungal isolate designated as AR.1 was subjected to molecular identification through amplification and sequencing of ITS genes according to the protocol of White et al. 69 . The ITS gene was amplified using a primer of ITS1 (5′ CTT GGT CAT TTA GAG GAA GTAA-3′) and ITS4 (5′ TCC TCC GCT TAT TGA TAT GC 3′). The PCR mixture containing the following: 0.5 mM MgCl 2 , PCR buffer, 2.5 U Taq polymerase (QIAGEN, GERMANTOWN, MD-20874, USA), 0.5 µM of each primer, 2.25 mM dNTP, extracted genomic DNA (5 ng). The run was achieved using DNA Engine Thermal Cycler (PTC-200, BIO-RAD, USA) and adjusted at 94 °C for three minutes followed by 30 cycles at 94 °C for a half minute, 55 °C for a half minute, 72 °C for one minute, and finally 72 °C for ten minutes. The PCR products were checked using agarose gel (1%) before being sequenced on GATC Biotech [A company using a DNA sequencer (ABI-3730xl) as a partner of Sigma Aldrich, Cairo, Egypt]. The obtained sequences were compared by the database deposited in GenBank by ClustalX 1.8 software package (http:// www. clust al. org/ clust al2) 70,71 . The Phylogenetic analysis was achieved using the neighbor-joining method (MEGA v6.1) software, with confidence tested by bootstrap analysis (1000 repeats).
Characterization of Se-NPs. Fourier transform infrared (FT-IR) (Cary-660 model) was used to detect the different functional groups in fungal biomass as well as in as-formed Se-NPs. In this analysis, 10 mg of synthesized Se-NPs was mixed with KBr and pressed to form a disk subjected to scanning at a wavelength in www.nature.com/scientificreports/ the ranges of 400-4000 cm -122 . The crystallographic structure of fungal mediated green synthesis of Se-NPs was assessed by X-ray diffraction (XRD, PANalytical-X'Pert-Pro-MRD) containing CuKα electrode as a source of X-ray (λ = 1.54 Å). The analysis was achieved at current and voltage of 30 mA and 40 kV respectively in the range of 2θ values of 10°-80°. The crystallite size was calculated based on XRD analysis by Debye-Scherrer's equation as follows: where K is a Scherrer constant which was 0.94, λ is the wavelength of the X-ray which was 1.54, β is the full width of the diffraction peak at a half maximum, and θ is the diffraction angle. The morphological characteristics (shape and size) were checked by transmission electron microscopy (TEM, JEOL, Ltd-1010, Tokyo, Japan). The green synthesized Se-NPs powder was suspended in H 2 O under ultrasonication and added a few drops on the TEM-carbon grid. The loaded grid was left to dry before being subjected to analysis 72,73 .
Dynamic light scattering (DLS) (Nano-ZS, Malvern Ltd, Malvern, UK) was used to investigate the size distribution in the colloidal solution. The synthesized Se-NPs were dispersed in a high pure solvent (MiliQ H 2 O) to prevent the appearance of shadow on the signal during scattering analysis. Moreover, the surface charge of synthesized Se-NPs was assessed using Zeta-sizer apparatus (Nano-ZS, Malvern, UK) 74 .
Antimicrobial activity. The activity of fungal-based Se-NPs as antimicrobial agents was investigated toward a group of pathogenic microbes including Escherichia coli ATCC8739, Pseudomonas aeruginosa ATCC9027 (Gram-negative bacteria), Bacillus subtilis ATCC6633, and Staphylococcus aureus ATCC6538 (Gram-positive bacteria). Also, the activity was assessed against a group of clinical Candida strains designated as C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis which were collected from Microbiology Laboratory, National Research Centre, Giza Egypt. The antimicrobial activity was assessed using the agar well diffusion method 75 . In this method, the selected bacterial and fungal strains were subculture on nutrient agar (Ready-prepared, Oxoid) and sabouraud dextrose agar plates (containing gL -1 : dextrose, 40; peptone, 10; agar, 15), respectively for overnight at 35 ± 2 °C. After that, a single colony from each organism was picked up and spread uniformly by sterilized swab on the surface of the Muller-Hinton agar plate (Ready prepared, Oxoid) followed by making four wells (0.6 mm in diameter) in each plate. These wells were filled with 100 µL of the prepared Se-NPs solution (400, 300, 200, 100, 50, 25, 12.5, and 6.25 µg mL -1 ). The solvent system (DMSO) was running with the experiment as control. The filled plates were incubated at 35 ± 2 °C for 24 h. At the end of incubation time, the results were recorded as a diameter of a clear zone formed around each well. The lowest Se-NPs concentration that inhibits microbial growth (MIC value) was determined. The experiment was conducted in triplicate.
Antioxidant activity. The antioxidant activity of fungal-mediated biosynthesis of Se-NPs was assessed by DPPH (2,2-diphenyl-1-picrylhydrazyl) method. In this method, various concentrations of biosynthesized Se-NPs (1.95-1000 µg mL -1 ) were prepared in high pure water (Milli-Q H 2 O). After that, one mL of prepared solution was added to a test tube containing one mL of DPPH (prepared in methanol) and 450 µL of Tris-HCl buffer (pH 7.4, 50 mM), mixed well before being incubated at 37 °C for a half-hour under shaking condition (100 rpm) in dark. Another set of experiments using ascorbic acid (positive control) was conducted under the same conditions /concentrations. Also, the negative control which was DPPH and Tris-HCl buffer in the absence of Se-NPs or ascorbic acid was running with the experiment under the same incubation conditions. At the end of the incubation period, the absorbance of the formed color was measured at 517 nm. The free radical scavenging percentages were calculated using the following Eqs. 6 : where Ab C and Ab T are the absorbances of the control (ascorbic acid) and treatment (Se-NPs), respectively.

Anticancer and biocompatibility test. A-Source of cell lines. The two cancer cells, designated as MCF7
(human breast cancer) and PC3 (prostate cancer cell), and two normal cells represented by Vero (monkey kidney epithelial cell) and WI38 (Human lung fibroblast) were purchased from the Holding Company for Biological Products and Vaccines (VACSERA), Cairo, Egypt.
B-MTT assay. The anticancer activity of Se-NPs against two cancer cells (MCF and PC3) and the biocompatibility test toward two normal cells (Vero and WI38) was assessed using the MTT assay method. In this method, each cell type was inoculated in 96-well tissue culture plates with intensity 1 × 10 5 cells/100 µL/well and incubated at 37 °C for 24 h in a 5% CO 2 incubator. Once the monolayer sheet was formed, it was rinsed twice with washing media and adding 100 µL of RPMI maintenance media with 2% serum. After that, the growing cells were treated with double-fold concentrations of Se-NPs (31.25-1000 μg mL -1 ) and incubated for 48 h. Three wells without Se-NPs were used as control. After the incubation period, the remaining media in each well was discarded and received 50 µL of MTT solution (5 mg mL -1 of phosphate buffer saline solution) and shaken thoroughly for 5 min before being incubated for 4 h at 37 °C. After the complete incubation period, the MTT solution was discarded, and adding 100 µL of DMSO (10%) was to dissolve the formed formazan crystal through shaking for 30 min. The absorbance of the formed color was measured at 570 nm by an ELIZA reader 76 . The morphological changes in the cells due to Se-NPs treatment were observed using inverted microscopy (Nikon, www.nature.com/scientificreports/ ECLIPSE Ts2, Shinjuku, Tokyo, Japan), whereas the cell viability percentages were calculated by the following equation: where Ab T and Ab C are the absorbances of treatment and control, respectively.
Larvicidal activity. The efficacy of Se-NPs in the killing of instar larvae of Aedes albopictus was investigated.
The larvae of A. albopictus mosquito have been obtained from Medical Entomology Centre, Giza, Egypt. The collected larvae were kept in plastic cups filled with deionized water and preserved under laboratory conditions. A mixture of yeast and dog food (1: 1 w/w) was used to feed the larvae. The experiment was conducted at 70% relative humidity, 30 °C, and photoperiod 12: 12 h (dark/light) conditions. The experiment was carried out according to the standard of WHO guidelines 77 . In this method, 25 healthy larvae from each instar (I, II, III, and IV) were incubated separately in a container containing 200 mL of tap water mixed with Se-NPs concentration (50,40,30,20, and 10 µg mL -1 ) for 48 h. The container containing tap water without Se-NPs was used as a control. The larvae were considered dead if they lost their ability to reach the tap water surface after disturbance of the container. The mortality percentages were calculated using the following equation: where A is the mortality in control and B is the mortality in treatment. The experiment was carried out in five replicates for each Se-NPs concentration.
Statistical analysis. The statistical package SPSS v17 was used to analyze the obtained data and represented by the means of three independent replicates. The t-test or ANOVA followed by the Tukey HSD test at p < 0.05 was used to measure the difference between treatments. The larvae mortality percentages were measured by probit analysis, with LC 50 and LC 90 calculated using Finney's method.
Ethics approval and consent to participate. "Our work complies with the institutional, national, and international guidelines and legislation. "

Conclusion
Among four endophytic fungal strains that colonized garlic roots, P. verhagenii was selected as the best Se-NPs producer. The selected strain was identified by traditional methods as well as sequencing of ITS genes. The synthesized Se-NPs were characterized by UV-Vis spectroscopy, FT-IR, XRD, TEM, DLS, and Zeta potential analyses. Antimicrobial activity, antioxidant, in-vitro cytotoxicity against cancer and normal cell lines, and larvicidal activity were investigated. Data showed varied inhibition zones due to treatment of pathogenic Gram-positive bacteria, Gram-negative, and unicellular fungi with different concentrations and MIC values in the ranges of 12.5-100 µg mL -1 . Free radical scavenging activity was investigated using the DPPH method compared with ascorbic acid. Se-NPs reveal varied DPPH-scavenging activity based on the concentrations to be in the ranges of 31-87%. In addition, Se-NPs target cancer cells, MCF7 and PC3 with low concentrations compared to normal cells, WI38 and Vero. This finding promotes their integration into cancer treatment without negligible effects on normal cells. Moreover, the as-formed Se-NPs can be used as a larvicidal agent for a biomedical insect, A. albopictus with mortality percentages of 51% for IV-instar larvae and 85% for I-instar larvae at a concentration of 50 µg mL -1 . The obtained data confirmed that the endophytic fungi possess a high potential to fabricate active Se-NPs which can integrate into various sectors.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The sequence in the current study was deposited in NCBI (GenBank) at https:// www. ncbi. nlm. nih. gov/ nucco re/ OP471 232. www.nature.com/scientificreports/